In the early part of the 20th century, vitamin B3 was identified as a component missing from the diet of pellagra patients. Supplementation with nicotinic acid, or niacin, ameliorated the symptoms of pellagra, and prevented the onset of this condition in areas where it was prevalent. The biochemical role of niacin was elucidated in the 1930s, when it was found to be critical for the biosynthesis of nicotinamide adenine dinucleotide (NAD), a compound essential for cellular respiration (Preiss, J.; Handler, P. Biosynthesis of Diphosphopyridine Nucleotide I. Identification of Intermediates J. Biol. Chem. 1958 233, 488-492; Preiss, J.; Handler, P. Biosynthesis of Diphosphopyridine Nucleotide II. Enzymatic Aspects J. Biol. Chem. 1958 233, 493-500). The precise role of NAD in cellular respiration is well understood. As glucose and fatty acids are oxidized, NAD can accept a hydride equivalent, which results in its reduction to NADH. NADH can donate a hydride equivalent, resulting in oxidation back to NAD. These reduction-oxidation cycles use NAD for the temporary storage of hydride ion, but they do not consume NAD. There are other enzymes that use NAD in a different manner, and for purposes not directly related to energy production. Poly-ADPribose polymerases (PARPs), ADPribose transferases (ARTs), and sirtuins all catalyze reactions that release nicotinamide from NAD. This reaction generates a significant amount of energy, similar to ATP hydrolysis. The reverse reaction does not occur readily, so NAD must be replenished by other mechanisms (Bogan, K. L.; Brenner, C. Nicotinic Acid, Nicotinamide, and Nicotinamide Riboside: A Molecular Evaluation of NAD+ Precursor Vitamins in Human Nutrition Annu. Rev. Nutr. 2008, 28, 115-130).
Niacin (or nicotinic acid (pyridine-3-carboxylic acid)), and its amide niacinamide (or nicotinamide (pyridine-3-carboxamide)) are converted to NAD in vivo. In mammals, niacinamide, rather than niacin, may be the major NAD precursor. The set of biosynthetic transformations from niacinamide to NAD is shown in FIG. 1. The rate limiting step for this pathway is the formation of the bond between niacinamide and 5-phosphoribose-1-pyrophosphate (PRPP), and it is catalyzed by nicotinamide phosphoribosyl transferase (NAMPT) (Revollo, J. R.; Grimm, A. A.; Imai, S.-I. J. Biol. Chem. 2004, 279, 50754-50763). The NAMPT pathway is thought to be the most efficient route known for nicotinamide recycling. Niacin enters into a similar set of transformations, but in a final step, the carboxylic acid must be converted to a carboxamide to produce NAD. The biosynthesis of NAD from niacin follows the Preiss-Handler pathway (FIG. 1).
In 1982, nicotinamide riboside (NR) was investigated as a NAD precursor in prokaryotes (Liu, G.; Foster, J.; Manlapaz-Ramos, R.; Loivera, B. M. “Nucleoside Salvage Pathway for NAD Biosynthesis in Salmonella typhimurium” J. Bacteriol. 1982, 152, 1111-1116). In contrast to niacin, exogenously supplied NR is hypothesized to bypass the first and most energy-consuming part of both the Preiss-Handler pathway and the NAMPT pathway (FIG. 1). Although NR appears to be a natural precursor for NAD, it likely contributes only a small amount to NAD biosynthesis owing to the apparent scarcity of NR in dietary sources. NR contains a high energy glycosidic bond that is spontaneously labile in aqueous solution, yielding nicotinamide and ribose decomposition products. This spontaneous reaction occurs over the course of hours or days depending on the exact ambient conditions, but it makes any naturally occurring NR difficult to keep in food sources, while nicotinic acid or nicotinamide are considerably more stable and easy to prepare and administer. NR has been reported to occur in milk (Bieganowski and Brenner (2004) Cell 117: 495-502) and beer, but the amounts typically present are probably too small to be nutritionally significant.
Currently, NR supplementation is limited by the available commercial supply. NR supplementation could represent a dietary alternative to niacin, with the advantage of being a more efficient NAD precursor. By taking advantage of a natural pathway to synthesize NAD while consuming less energy, NR could offer benefits for human health. Cells are constantly subject to damage by normal environmental factors, and they have evolved repair mechanisms to continuously reverse this damage. The repair mechanisms consume NAD by scission of the high energy glycosidic linkage to produce species such as poly-ADPribose and ADP-ribosylated proteins. In severely damaged cells, energy stores are not sufficient to produce the NAD necessary to maintain homeostasis, and the damage becomes irreversible. Therefore, an energy-rich NAD precursor such as NR may be able to address cell and tissue damage at the molecular level.
NR is difficult to isolate from natural sources, so it is almost always produced by chemical synthesis. The first chemical synthesis was accomplished by Todd and co-workers in 1957 (Haynes, L. J.; Hughes, N. A.; Kenner, G. W.; Todd, A. J. Chem. Soc. 1957, 3727-3732). This group produced NR chloride as α mixture of a and β anomers about the glycosidic linkage in an approximately 1:4 ratio. The product was described as a hygroscopic oil that could not be crystallized. Other investigators who isolated NR chloride from biochemical sources also described it as a hygroscopic oil (Schlenk, F. “Nicotinamide Nucleoside” Natunviss. 1940, 28, 46-47; Gingrich, W.; Schlenk, F. “Codehydrogenase I and Other Pyridinium Compounds as V-Factor for Hemophilus Influenzae and H. Parainfluenzae” J. Bacteriol. 1944, 47, 535-550). Significantly, biochemical syntheses should have produced only the natural β-anomer, though the exact stereochemical arrangement was not determined. Later reports confirmed the hygroscopic, amorphous nature of NR chloride (Jarman, M.; Ross, W. C. J. J. Chem. Soc. C, 1969, 199-203; and Atkinson, M. R.; Morton, R. K.; Naylor, R. Synthesis of Glycosylpyridinium Compounds from Glycosylamines and from Glycosyl Halides J. Chem Soc. 1965, 610-615). Other groups investigated alternative NR anions. One synthesis described the anomerically pure NR bromide salt as crystalline, but the product was not adequately described to ascertain whether the material was truly crystalline or merely an amorphous solid (Lee, J.; Churchill, H.; Choi, W.-B.; Lynch, J. E.; Roberts, F. E.; Volante, R. P.; Reider, P. J. “A chemical synthesis of nicotinamide adenine dinucleotide (NAD+)” Chem. Commun. 1999, 729-730). Subsequently, other NR salts were prepared and solids were obtained, though they were never described as crystalline (Tanimori, S.; Ohta, T.; Kirihata, M. An Efficient Chemical Synthesis of Nicotinamide Riboside (NAR) and Analogues Bioorg. Med. Chem. Lett. 2002, 12, 1135-1137; Franchetti, P.; Pasqualini, M.; Petrelli, R.; Ricciutelli, M.; Vita, P.; Cappellacci, L. Bioorg. Med. Chem. Lett. 2004, 14, 4655-4658; Yang, T.; Chan, N. Y.-K.; Sauve, A. A. J. Med. Chem. 2007, 50, 6458-6461).
Previously described NR salt preparations are amorphous NR and extremely hygroscopic, becoming sticky solids within seconds or minutes and collapsing to oils within hours at ambient temperature and humidity. Maintaining the amorphous salts as solids required storing them under a dry atmosphere, or keeping them frozen at approximately −20° C. Importantly, the oily mixtures decomposed significantly over the course of one day at ambient temperature. This property presents a major challenge for isolating and handling NR salts. It also makes it difficult to specify the purity of an NR preparation, because some handling under ambient conditions is inevitable during analysis or use. Ease of handling and purity are important parameters for a substance that might be manufactured for human consumption. These are also important considerations for a substance that will be used for any subsequent purpose, for example as a synthetic intermediate for another chemical transformation, as a biochemical reagent, as an analytical standard, or for any other use where chemical purity and stability are desired.
Furthermore, while several of the previously described preparations of anomerically pure NR salt crystals have been bromide rather than chloride salts, bromide salts may be unnecessarily toxic or otherwise undesireable as a pharmaceutical salt form compared to corresponding chloride salts. For example, bromide compounds, especially potassium bromide, was frequently used as sedatives in the 19th and early 20th century, but their use in over-the-counter sedatives and headache remedies (such as Bromo-Seltzer) ended in the United States in 1975, when bromides were withdrawn due to chronic toxicity. Doses of 0.5-1 gram per day of bromide can lead to bromism, a syndrome with multiple neurological symptoms and skin eruptions (see Olson, Kent R. (1 Nov. 2003). Poisoning & drug overdose (4th ed.) Appleton & Lange. pp. 140-141). In contrast, chloride is considered a “first class” pharmaceutical salt-former that can be used more or less without restriction as it represents a physiologically ubiquitous ion, and, indeed, healthy adults are even encouraged to consume 2.3 grams of chloride each day to replace the amount lost daily on average through sweat and to achieve a diet that provides sufficient amounts of other essential nutrients (see, Saal, C.; Becker, A. Eur J Pharm Sci 2013, 49(4), 614-623; and Institute of Medicine of the National Academies, 2013, Dietary reference intakes: water, potassium, sodium, chloride, and sulfate, from the Institute of Medicine of the National Academies:<http://www.iom.edu/Reports/2004/Dietary-Reference-Intakes-Water-Potassium-Sodium-Chloride-and-Sulfate. Therefore chloride pharmaceutical salts are generally safer than corresponding bromide salt forms, particularly for pharmaceutical salts that require relatively high dosages.
Accordingly, there is a need for a chemically pure and stable form of a pharmaceutically acceptable NR salt such as nicotinamide riboside chloride, as well as for corresponding methods for its synthesis and efficient preparation on a large scale.